Blowflies & Nicotine: an Entomotoxicology Study Paola A. Magni1, Marco Pazzi2, Eugenio Alladio2, Marco Vincenti2, Marco Brandimarte2, Ian R. Dadour3

1 School of Veterinary & Life Science, Murdoch University WA; 2 Department of Chemistry, University of Turin, Italy 3 Program in Forensic Anthropology, Department of Anatomy & Neurobiology, Boston University School of Medicine

Introduction

“Entomotoxicology” is the term used to describe the science involving the combination of entomology and toxicology. Entomotoxicology examines the adverse effects of chemicals on insects feeding on the remains of humans and other animals [1]. Toxicological substances (“drugs”) present in remains can also accumulate in necrophagous insects. Many of these drugs affect insects, altering their rate of development and survival [2]. In a forensic context, the identification of drugs in necrophagous insects may help determine the cause of death because on decomposed tissues the common toxicological analyses generally provides poor sensitivity and may yield erroneous results [3,4].

At present, only a modest number of substances and insect species/life instars have been studied and many early studies utilized analytical procedures which are now obsolete with little or no validation [5]. This is the first entomotoxicological study concerning the detection, analytical quantification and the effect of nicotine on any necrophagous entomofauna.

Nicotine is a volatile and water-soluble alkaloid present in the leaves and stems of the plants of Nicotiana species (Solanales: Solanaceae), which includes N. tabacum L., the tobacco plant [6]. The tobacco plant was considered to have therapeutic properties able to treat a wide range of disorders. However, several cases of patients treated with tobacco showed fatal or poisonous exitus [7]. Nowadays tobacco is less used in medicine, but nicotine can be found in tobacco products, such as cigarettes, cigars, pipe and chewing tobacco, and refill solutions for electronic cigarettes. Furthermore, nicotine is present in various formulations of nicotine replacement therapy, such as nicotine transdermal patches, nasal sprays, inhalators, gums, sublingual tablets and lozenges [8]. In some countries, nicotine is also present in toothpastes to whiten teeth [9]. Finally, nicotine is also used as a synergist in insecticides [10].

Nicotine has acute toxicity; it is considered one of the most deadly poisons known to man and, at the same time, it’s extremely easy to come in contact with during normal daily life (e.g. buying smoking products) [11]. Nicotine can be readily absorbed across the epithelium of the lung, the nose, and through the skin and mucosae, regardless of the mode of administration [11]. Therefore, there is a potential for poisoning from ingestion, injection, inhalation, skin and rectum absorption of nicotine from nicotine-containing products, including insecticides and tobacco products [12]. The literature reports a number of accidental/sudden, suicidal and homicidal cases whereby nicotine (alone or mixed with other drugs) was used [12].

Nicotine and its metabolites (e.g. cotinine, the major metabolite of nicotine) can accumulate in human hair and nails [13]. In a nicotine overdose situation, the toxicological examinations will be focused on the presence of nicotine in the liver, while nicotine metabolites would provide only accessory information [13]. This research describes the development and validation of a suitable analytical method, based on GC-MS, to detect nicotine in larvae, pupae, empty puparia and adults of blowfly Calliphora vomitoria L. (Diptera: Calliphoridae). Furthermore, the effects on the blowfly survival and growth rate were examined when reared on substrates spiked with three concentrations of nicotine, sufficient to cause death in humans [14].

Experimental Design

C. vomitoria were reared in the insectary of the Entomotoxicology Lab at University of Turin from already established and maintained colonies. Adult flies were fed daily with water and sugar cubes ad libidum. Five days after emergence flies were provided with fresh beef liver to allow the ovaries to develop. After 10 days fresh beef liver was placed in the cages on water moistened paper on small plastic trays in order to allow females to oviposit.

Fly eggs were then moved on beef liver spiked with different amounts of nicotine (T1=2 ng/mg, T2=4 ng/mg, T3=6 ng/mg ). Another liver was used as control. The appropriate nicotine spiking concentrations were selected based on the concentrations that would most likely cause death in a human [14].

Two samples, one consisting of 30 individuals and another amounting to 1 g from each treatment were collected when C. vomitoria reached the second (L2), third (L3), post-feeding (PF) pupal (P) and adult (A) instars. Empty puparia (EP) were also collected. Each sample of 30 individuals was used for morphological analyses. Each sample weighing 1 g from each of the instars was analysed to detect nicotine.

The validation of the method for GC-MS for nicotine detection was performed according to ISO/IEC 17025 requirements and ICH guidelines [15]. The validation protocol included the quantitative determination of nicotine in larvae, P and EP: specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), extraction recovery, repeatability and carry over were determined (Table 3).

Nicotine and cotinine concentration in larvae and pupae as well as their respective lengths in different treatments were analysed by one-way ANOVA and Tukey test. Pupation and eclosion rate were analysed by a one-way ANOVA and Pearson’s Chi-squared test. The level of significance was set at P < 0.05. Calculations were performed using IBM SPSS Statistics 22 statistical package.

Results & Conclusions

1 - The GC-MS method can detect both nicotine and its metabolite cotinine in C. vomitoria immatures (Table 1);

2 – The presence of nicotine at the 3 scheduled concentrations in the food substrate did not modify the developmental time of C. vomitoria (Table 2);

3 - During the pupation period larvae exposed to nicotine died dependent on the concentration of nicotine in the substrate (Table 2);

4 - The resultant lengths of larvae and pupae exposed to 4 ng/mg and 6 ng/mg concentrations of nicotine were significantly shorter than the control (Fig. 2).

CTRL CTRL CTRL CTRLT1 T1* T1* T1*T2 T2* T2* T2*T3 T3* T3* T3*0

2

4

6

8

10

12

14

16

18

20

60h(L2) 120h(L3) 168h(PF) 216h(P)

MeanlengthofC

.vom

itoria

immatureinstars(mm±S.E.)

Hoursofexposure(Instar)

CTRL,0ng/mgnico:ne T1,2ng/mgnico:ne T2,4ng/mgnico:ne T3,6ng/mgnico:ne

Parameter Value

Nicotine Cotinine Coefficient of linearity, R2 0.9954 0.9930

Limit of detection, LOD 0.13 ng/mg 0.38 ng/mg

Limit of quantification, LOQ 0.43 ng/mg 1.2 ng/mg

Extraction recovery at 2 ng/mg concentration 71.11 % NC

Extraction recovery at 6 ng/mg concentration 69.23 % NC

CV % at 2 ng/mg concentration 14.65 % NC

CV % at 6 ng/mg concentration 15.80 % NC

Table 3 – Parameters calculated for nicotine and cotinine. NC = not calculated.

Treatment Control (C) T1 T2 T3

Amount of nicotine spiked with liver 0 ng/mg 2 ng/mg 4 ng/mg 6 ng/mg

Quantification (ng/mg ± S.E.) Nicotine Cotinine Nicotine Cotinine Nicotine Cotinine Nicotine Cotinine

Life

inst

ar

L2 < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD

L3 < LOD < LOD 0.77 ± 0.12 (C, T3) < LOD 1.53 ± 0.23

(C, T3) < LOD 3.08 ± 0.46 (C, T1,T2) < LOD

PF < LOD < LOD 0.98 ± 0.15 (C, T2) < LOD 0.53 ± 0.08

(C, T1, T3) < LOD 1.06 ± 0.16 (C, T2) < LOD

P < LOD < LOD 0.39 ± 0.06 (C, T3)

< LOD (T2, T3)

0.50 ± 0.08 (C, T3)

1.11 ± 0.17 (C, T1, T3)

0.89 ± 0.13 (C, T1,T2)

1.84 ± 0.25 (C, T1, T2)

EP < LOD < LOD 0.82 ± 0.07 (C, T3)

0.90 ± 0.01 (C, T3)

1.82 ± 0.27 (C, T3)

1.41 ± 0.12 (C, T3)

3.29 ± 0.46 (C, T1, T2)

2.78 ± 0.29 (CT, T1, T2)

A < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD

Table 1 – Nicotine and cotinine quantification (ng/mg ± S.E.) in C. vomitoria (L2=second instar, L3=third instar, PF=post-feeding instar, P=pupa instar, EP=empty puparium, A=adult instar) through GC-MS analysis. Quantification was calculated using 3 replicates. Nicotine LOD=0.13 ng/mg; Cotinine LOD=0.38 ng/mg. The groups indicated in brackets (i.e. C, T1, T2, T3) are the ones whose results proved significantly different (P<0.05) from the group indicated in the corresponding column.

Treatment Control (C) T1 T2 T3

Larvae third instar N= 100 100 100 100

Time (h) from oviposition to pupation 163.82 ± 1.01 164.52 ± 1.21 164.62 ± 1.30 163.98 ± 1.31

Larvae dead prior to pupation 2 2 3 5

Pupae 98 98 97 95

Pupae % 98% 98% 97% 95%

Pupae N= 98 98 97 95

Time (h) from oviposition to eclosion 468.92 ± 1.25 470.92 ± 1.05 470.04 ± 1.24 469.08 ± 1.51

Not emerged adults 8 15 20 33

Survival 90 (T2, T3) 83 (T3) 77 (C, T3) 62 (C, T1, T2)

Survival % 92% 84.70% 79.38% 65.26%

Table 2 – Time (hour mean ± S.E.) from oviposition to pupation and to eclosion of C. vomitoria larvae, which were exposed to either liver containing different amount of nicotine, or to the control liver. The table shows also the number of larvae dead prior to pupation, the number of not emerged adults, and the number of survivals. The groups indicated in brackets (i.e. C, T1, T2, T3) are the ones whose results proved significantly different (P<0.05) from the group indicated in the corresponding column.

Fig. 2 – Mean length (mm ± S.E.) of C. vomitoria immature instars accordingly to treatment type, time of exposure and developmental instar. (*) indicates significant difference compared to the control group (P<0.05).

Fig. 1 – Background subtracted mass spectrum of nicotine (a) and cotinine (b) obtained with electronic impact (EI) ionization. The mass-to-charge ratio (m/z) for nicotine is 162, and for cotinine is 176.

References [1] Byrd JH, Peace MR. Entomotoxicology: Drugs, Toxins, and Insects. In: Kobilinsky L. Forensic Chemistry Handbook 2011:483-99. [2] Carvahlo LML. Toxicology and forensic entomology. In: Amendt et al. Current Concepts in Forensic Entomology: Springer Science; 2010:163–78. [3] Magni et al. Entomologia Forense. Gli insetti nelle indagini giudiziarie e medico-legali: Ed. Minerva Medica; 2008. [4] Haskell NH, Williams RE. Entomology & Death: A Procedural Guide. 2 ed: Forensic Entomology Partners, Clemenson, SC, USA; 2009. [5] Gosselin et al.. Entomotoxicology, experimental set-up and interpretation for forensic toxicologists. Forensic Sci Int 2011;208:1-9. [6] Schep et al. Nicotinic plant poisoning. Clin Toxicol 2009;47:771-81. [7] Charlton A. Medicinal uses of tobacco in history. J R Soc Med. 2004;97:292-6. [8] Benowitz et al. Nicotine chemistry, metabolism, kinetics and biomarkers. Handbook of Experimental Pharmacology 2009:29-60. [9] Agrawal SS, Ray RS. Nicotine contents in some commonly used toothpastes and toothpowders: a present scenario. J Toxicol. 2012;2012:1-11. [10] Hayes WJ. Pesticides studied in man. Baltimore: Lippincott Williams & Wilkins; 1982. [11] Faulkner JM. Nicotine poisoning by absorption through the skin. J Am Med Assoc. 1933;100:1664-5. [12] McNally WD. A report of seven cases of nicotine poisoning. J Lab Clin Med. 1922;8:83-5. [13] Crooks PA, Dwoskin LP. Contribution of CNS nicotine metabolites to the neuropharmacological effects of nicotine and tobacco smoking. Biochem Pharmacol. 1997;54:743-53. [14] Mayer B. How much nicotine kills a human? Tracing back the generally accepted lethal dose to dubious self-experiments in the nineteenth century. Arch Tox 2014;88:5-7. [15] AA.VV. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Validation of analytical procedures: text and methodology Q2 (R1). 2005.

This research has been recently published as:

Magni et al., Development and validation of a GC–MS method for nicotine detection in Calliphora vomitoria (L.) (Diptera: Calliphoridae). Forensic Sci Int 2016

Magni, P., Pazzi, M., Alladio, E., Vincenti, M., Brandimarte, M. and Dadour, I. (2016) Blowflies & nicotine: an entomotoxicology study. In: AAFS 68th Annual Scientific Meeting, 22 - 27 February, Las Vegas, Nev, USA